CN1094978C - Process for biotransformation of colchicinoid compounds into corresponding 3-glycosyl derivatives - Google Patents

Abstract

Colchicinoid compounds are transformed into the corresponding 3-O-glycosyl derivatives by means of Bacillus megaterium strains.

Description

Method for bioconverting colchicine compounds into corresponding 3-glycosyl derivatives

The present invention relates to a method for the biotransformation of colchicine compounds into corresponding 3-O-glycosyl derivatives using selected microbial strains. The method of the present invention can produce colchicine compounds glycosylated only at C-3 of aromatic ring A from colchicine, thiocolchicine or derivatives thereof with high yield and high purity.

Colchicines glycosylated at the C-3 position of the phenyl ring are of great pharmacological importance because they are very potent or useful for the preparation of new drugs.

Specifically, thiocolchicoside (3-O-glucosylthiocolchicine) is a very important active ingredient in the pharmaceutical field, mainly used for the treatment of diseases of the musculoskeletal system, and as a Raw materials for the preparation of new anti-neoplastic, immunosuppressive, anti-psoriatic and anti-inflammatory drugs.

Previously, many attempts have been made to prepare 3-glycosylcolchicines by chemical reaction or biotransformation.

The chemical route consists of a series of complex, nonspecific reactions that result in a mixture of glycosylated derivatives, some of which are inactive. Thus, the conversion yields very low yields of efficient products specifically glycosylated at the C-3 position of the aromatic ring.

The biological pathway actually involves the biotransformation of thiocolchicine to derivatives monoglycosylated at the C-2 and C-3 aromatic rings by culturing Centella Asiatica; thus, the The selectivity is not high, and the yield and yield are very low (Solet, J.M. et al., Phytochemistry 33, 4, 817-820, 1993).

Other attempts to biotransform colchicines have demethylated only the methoxy groups attached to the aromatic rings (C-2 and C-3 positions), however, the yields and yields of these methods are generally poor. low, and poor regional selectivity.

Therefore, Hufford C.D. et al. (Journal of Pharmaceutical Sciences, 68, 10, 1239-1242, 1979) used Streptomyces griseus and/or Streptomyces spectiformis, Bellet.P. et al. (GB-923421, 1959) used different strains of Streptomyces and Other species of bacteria and fungi have attempted to convert colchicine and its derivatives into the corresponding 3-demethylated derivatives. The results of these known methods confirm the above descriptions concerning the non-stereoselectivity involved in microbial enzymes, for example at the C-2, C-3 or C-10 position of the alkaloid molecule. Furthermore, the production levels of the catalytic system are very low due to low conversion yields, reduced concentrations of usable substrates and frequent degradation of tropolone.

Recently, Poulev et al. (J. Ferment. Bioeng. 79, 1, 33-38, 1995) achieved specific biotransformation of bacterial microorganisms, but the yield and yield were still very low.

Enzymatic activities from microorganisms similar to the above-mentioned microorganisms (Streptomyces, Bacillus) have been applied to biotransform other compounds, such as maytansinoids (US Patent, 4361650; Izawa, M. et al., J. Antibiotics, 34, 12 , 1587-1590, 1981). In this case, too, the catalytic reaction consists entirely of demethylation reactions, and the conversion and yield are low.

The glycosyltransferase activity of an α-amylase from Bacillus megaterium has been documented (Brum, P.J. et al., Starch 43, 8, 319-323, 1991); Body specificity is very high. Cyclodextrin-glucosyltransferase produced by the same microorganism can catalyze the α-1,4 - Transglucosylation. In addition, in this biotransformation, the acceptor of the transferase reaction is the substrate sugar moiety (Darise, M. et al., Agric. Biol. Chem., 48, 10, 2483-2488, 1984). Cyclodextrin-glycosyltransferases were previously used to prepare cyclodextrins G6, G7 and G8 from starch (Kitahata, S., Okada, S., Agricultural Biochemistry 38, 12, 2413-2417, 1974).

These examples demonstrate the high substrate specificity of the glycosyltransferase activity expressed by Bacillus megaterium, which is only associated with sugar acceptors and thus does not involve any secondary metabolites such as colchicines with different complex molecular structures reaction. In fact, there are no known examples of the use of said microorganisms for the enzymatic conversion of colchicines into 3-glycosyl derivatives.

It has now been found that Bacillus megaterium strains that grow in the presence of high concentrations of colchicine and thiocolchicine have very high biotransformation activity, converting colchicine-like substrates into 3 Glycosylated colchicine. The conversion takes place in a short time and in surprisingly high yields.

其中R₁是O-糖苷基，R₂是氢或C₁-C₇酰基，R₃是C₁-C₆烷氧基或C₁-C₆硫代烷基，该方法包括用巨大芽孢杆菌生物转化其中R₁是OH或甲氧基的化合物。Therefore, the present invention relates to a process for the preparation of 3-O-glycosylcolchicine compounds of formula (I)

Wherein R ₁ is O-glycosidic group, R ₂ is hydrogen or C ₁ -C ₇ acyl, R ₃ is C ₁ -C ₆ alkoxy or C ₁ -C ₆ thioalkyl, the method comprises using Bacillus megaterium Biotransform compounds in which _R is OH or methoxy.

Bacillus megaterium is a Gram-positive spore-producing bacterium greater than 1.0 μm in cell diameter; grows aerobically in many media; is positive for catalase; hydrolyzes gelatin.

As demonstrated by growth assays and microscopic analysis, the Bacillus megaterium strains used in the present invention grow satisfactorily and also at high concentrations of colchicine and/or thiocolchicine (higher than 3 g/l). can survive.

It has been proved that the same genus such as Bacillus cereus is difficult to grow when the substrate concentration is 1.5g/l (15-25% of the control absorption value), and it is more significant when the concentration is 3g/l, and there is a significant autolysis. In contrast, selected B. megaterium cultures reached very high levels of growth (2-fold or 3-fold) compared to B. cereus at the above mentioned concentrations.

The high selectivity and efficiency of the biotransformation is surprising and extraordinary, with yield levels of 80-100%, usually about 90-95%.

Furthermore, the microorganisms used in the biotransformation are capable of maintaining catalytic activity durably, even in repeated culture steps, thus providing specific biotransformation in both fed-batch and continuous culture processes. Therefore, the productivity and reproducibility of the method are high.

In addition to the remarkable productivity, the remarkable reaction regioselectivity ensures the high quality and purity of the obtained product, thus, the product can be provided in 100% purity, requiring only simple subsequent processing.

Another important advantage is that the purification and recovery steps of the product are reduced, the method is more economical and simple, and it is safer to use.

The operation process of screening the bacterial strains that can be used for the inventive method comprises:

A) Starting from natural sources or collected strains, B. megaterium were selected for growth in the presence of high concentrations of the colchicine substrate.

B) The isolates from A) were screened to demonstrate the conversion activity of colchicine into the corresponding 3-O-glycosyl derivative using a biotransformation assay on a specific substrate (applied in increasing concentrations).

C) Microbiological characteristics of selected strains in B).

D) Using B) target-specific screening of bacterial populations to gradually increase biotransformation yields.

E) Study and optimize standard fermentation parameters to optimize bioconversion.

F) Research and optimization of transformation methods for high productivity cultures to ensure a stable and uniform inoculum for industrial scale production.

G) Gradually scale up the process with fermenter, batch, fed-batch and continuous processing.

H) Workup and optimization methods for subsequent processing and recovery of products.

Specifically, by adding organic nitrogen (peptone, yeast extract, meat extract, asparagine, etc.) and carbon sources (glycerol, starch, maltose, glucose, etc.) 8. Preferably 6-7, the collected cultures obtained from strain collections or soil samples from various sources are screened for use in the microorganisms of the present invention. The culture temperature is 20-45°C, preferably 28-40°C.

Toxicity in the presence of The ability of the culture to grow at the concentration of the colchicine substrate to be converted.

Colonies capable of growing under the conditions described were aseptically removed and plated on different agarization media to determine their uniformity of purification and growth.

The media used for transformation cultures are typical microbial substrates on different agar media containing organic nitrogen (ptone, yeast extract, tryptone, meat extract, etc.), carbon sources (glycerol, maltose, glucose, etc.) , at a pH of 5-8, preferably 6-7, collected cultures that can be obtained from strain collections or soil samples from various sources are screened for microorganisms that can be used in the present invention. The culture temperature is 20-45°C, preferably 28-40°C.

The selected microorganisms are then tested for their ability to grow in submerged culture in the presence of the colchicoids and for their ability to convert the colchicoids to the corresponding 3-glycosyl derivatives.

The assay was done in a 100ml flask containing 20ml of liquid medium containing different medium preparations containing one or more organic nitrogen (yeast extract, peptone, tryptone, casein hydrolyzate, meat extract, corn steep liquor, etc.), one or more carbon sources (glucose, glycerol, starch, sucrose, etc.), inorganic phosphorus and nitrogen sources, and various ions (K ⁺ , Na ⁺ , Mg ⁺⁺ , Ca ⁺⁺ , Fe ⁺⁺ , Mn ⁺⁺ etc.) inorganic salts.

If necessary, conventional mutagenesis techniques (irradiation with UV rays, etc.) can be used to mutagenize the cultured samples to induce mutants having specific biotransformation activity, which can be evaluated by the above-mentioned method.

Culture samples in each biotransformation assay were analyzed by TLC or HPLC chromatography to assess the ability to produce 3-glycosyl derivatives.

The ability of the selected microorganisms to convert the colchicine-like substrates into the corresponding 3-glycosyl derivatives was confirmed using a biotransformation assay at a 300 ml scale using the same culture broth as the screening procedure.

Positively reacting microorganisms were used in experiments to optimize biotransformation in different culture broths on a 300ml scale. The main culture and fermentation parameters studied are: organic nitrogen source, carbon source, inorganic salt, temperature, agitation and aeration, pH, culture time, inoculation rate, subculture steps, and time for adding substrate to be transformed. The selected bacterial microorganisms capable of effecting the biotransformation of the present invention can be grown in solid and liquid media containing one or more sources of organic nitrogen, preferably yeast extract, meat extract, peptone, tryptone, Casein hydrolyzate, corn steep liquor, etc. Carbon sources for growth and biotransformation are glucose, fructose, sugar, glycerol, malt extract, etc., preferably glucose, fructose and glycerol. The medium also contains inorganic phosphorus sources and salts such as K ⁺ , Na ⁺ , Mg ⁺⁺ , NH ₄ ⁺ and the like.

The selected microorganisms can grow at a temperature of 20-45°C, preferably 28-40°C, and a pH of 5-8, preferably 6-7. Under the same conditions, the selected microorganism is capable of converting colchicines into the corresponding 3-glycosyl derivatives. The transformation takes place in submerged culture in the flask on a rotary shaker while stirring at 150-250 rpm.

Because of the specific kinetics involved in biotransformation with respect to microbial growth, optimal conditions for biotransformation are the same as for optimal growth. Therefore, media for promoting good growth of microorganisms, such as those based on the above-mentioned organic and inorganic components, also have good activity for the biotransformation of the substrates involved. The latter is added to the culture during the initial fermentation step.

The biotransformation of the present invention is based on enzymatic conversion and starts and continues during the exponential growth phase; the conversion of the 3-glycosyl derivative reaches a maximum level (very high: up to 95-100%) within the first 24-30 hours. The regioselectivity of the biotransformation is absolute: no 2-glycosyl derivatives are present. The resulting products are all extracellular products.

The biotransformation of the present invention can be scaled up to the fermenter level, keeping the culture conditions constant, especially with regard to medium, temperature and duration of time. For good growth, a sufficient level of agitation aeration is very important, in particular, aeration levels of 1-2 liters air/liter culture/minute (vvm), preferably 1.5-2 vvm are required.

After separation of the biomatrix from the liquid fraction by centrifugation and recovery of the supernatant, or microfiltration and osmotic recovery, the resulting bioconversion product is extracted from the culture broth. For optimal recovery of product, the culture is treated with alcohol.

Purification and recovery of the biotransformation product is accomplished by chromatographic techniques with separation on absorbent resins and elution with an alcohol, preferably methanol. The product-containing hydrogen methanolic solution is then further passivated with a lipophilic organic solvent, preferably dichloromethane. After further treatment with a mixture of alcohol and organic solvent, the pure product is obtained from the resulting alcoholic solution by crystallization.

The biotransformation method of the present invention is specific for substrates containing tropolone groups, and can be used for many colchicine compounds such as colchicine, thiocolchicine, 3-demethylcolchicine thiocolchicine, 3-demethylthiocolchicine, N-deacetylthiocolchicine and various other substituted colchicines.

Bacillus megaterium does not glycosylate other natural compounds that do not contain tropolone.

Glucose can be replaced by other sugars such as fructose or galactose without loss of glycosyltransferase activity.

The following examples disclose the present invention in more detail.

Example 1

Aliquots of Bacillus megaterium cultures isolated from agricultural soil were resuspended in 20 ml sterile saline and then serially diluted with a dilution factor of 1:10000000. The suspensions of different dilutions were spread on LB-agar medium, and colchicine or thiocolchicine (see table) were added with a final concentration of 2 g/l on the LB-agar. Cultures were grown at 28°C for 3 days in the dark. Colonies grown on selective media plus colchicines were isolated and purified by plating on non-selective media; the samples were incubated as above, but for a shorter period of time (24 hours). Subsequently, the culture was transferred to the same agar medium in the assay tube and incubated for 24 hours as above.

Aliquots of cultures selected as described above were used to inoculate 100 ml Erlenmeyer flasks containing 20 ml ST medium supplemented with colchicine or thiocolchicine at a final concentration of 0.4 mg/ml. The culture was grown overnight at 28°C on a rotary shaker at 200 rpm.

The conversion of the colchicine substrate was detected by TLC on silica gel, eluting the system with acetone:ethyl acetate:water 5:4:1, and analyzing the culture broth every 3-4 hours.

After 4 days of incubation, culture samples demonstrating catalytic activity of the 3-glycosyl derivatives were replicated onto the plates by serial dilution as described above and used to prepare new inoculum in test tubes. Biotransformation assays in flasks were repeated under the same conditions as above, but with significantly higher final concentrations of colchicine and thiocolchicine (1 mg/ml). The most active culture (substrate conversion equal to or higher than 80%) was used to prepare the inoculum as described in Example 3 using cryotubes.

surface

Medium composition 1) LB-agar (sterilization: 121℃×20')-pH7 tryptone 10g/l yeast extract 5g/l sodium chloride ST 10g/l agar (Agar Agar) 5 g Sterilizer: 121 ℃ × 20 ') -Ph7 glucose 20g/L glycerin 10g/L 胨 15g/L yeast extract 5g/L sodium chloride 3g/L ammonium chloride 3g/L potassium di potassium 8g/L phosphate two Potassium Hydrogen 3g/l Magnesium Sulphate Heptahydrate 0.5g/l

Example 2

The procedure described in Example 1 was repeated, starting from a culture of Bacillus megaterium from the following collection of strains (German Collection of Microorganisms, Braunschweig, Germany): DSM90, 509, 322, 333, 1667, 1670, 1671 .

DSM 90

DSM 509

DSM 322

DSM 333

DSM 1667

DSM 1670

DSM 1671

In the liquid culture medium, the culture selected in Example 1 and added with thiocolchicine (1mg/ml) was cultivated for 4 days: TLC analysis detected whether the conversion of the substrate to thiocolchicine occurred, Transformation rates ranged from 50% (strain DSM1671) to 70% (strain DSM90), to 80% and higher (strains DSM333, DSM509, DSM1667, DSM1670).

Example 3

A 100 ml Erlenmeyer flask containing 20 ml ST broth was inoculated with an aliquot of the culture from the tube screened in the above example.

Broth cultures were grown overnight at 30°C on a rotary shaker at 200 rpm. After culturing, a sterile solution of glycerol at a final concentration of 20% was added to the culture. The culture was then dispensed into 2 ml cryogenic tubes and immediately immersed in liquid nitrogen.

After several days, 10% of the culture was rapidly thawed at 37°C. Each cryogenic tube was used to inoculate a 100ml Erlenmeyer flask containing 20ml ST broth. The tubes were then incubated overnight at 28°C at 200 rpm (preincubation). After cultivation, each 2 ml of the preculture was aseptically transferred to 20 ml of fresh ST medium, at which point colchicine or thiocolchicine was added to a final concentration of 1 g/l. The biotransformation was completed and detected according to the conditions described in Example 1. The analysis showed that the conversion of the substrate to the 3-glycosyl derivative occurred in the aforementioned amounts (80% and higher), thus demonstrating the catalytic stability of the frozen cultures.

Parallel controls of broth cultures, plated on LB agar immediately after thawing, demonstrate viability, homogeneity and purity of frozen cultures.

Example 4

An aliquot of the culture in the cryotube was thawed and used to inoculate a 300ml Erlenmeyer flask containing 50ml ST medium (preculture). After culturing overnight at 30°C and 250 rpm, 5 ml of the preculture was transferred to 50 ml of the same medium supplemented with a final concentration of 1 g/l colchicine. The culture was grown for 2 days under the same conditions as above. Samples were taken every 4 hours to assess growth levels (measurement of absorbance at 600 nm), colchicine production (YLC and HPLC), sterility (on LB agar) and for microscopic morphology.

For HPLC analysis, 9 ml methanol was added to 1 ml culture broth and centrifuged at 13000 rpm for 2 minutes. The content of colchicoside in the supernatant was analyzed by reverse-phase HPLC using a water:acetonitrile 80:20 elution system.

HPLC analysis proved that the conversion of colchicine to colchicoside was synchronized with the growth. After approximately 26 hours of incubation, biotransformation was complete.

The final yield of colchicoside is 80%-85%.

Example 5

The procedure in Example 4 was repeated, adding thiocolchicine to the culture instead of colchicine at the same final concentration (1 g/l).

The growth and production responses of the cultures were similar to those obtained with colchicine, with about 90% yield of thiocolchicosides.

Example 6

The procedure in Example 4 was repeated, adding 3-demethylthiocolchicine to the culture instead of colchicine at the same final concentration (1 g/l).

The growth and production responses of the cultures were similar to those obtained above, with about 90% yield of thiocolchicosides.

Example 7

The procedure in Example 4 was repeated, adding N-formylthiocolchicine to the culture instead of colchicine at the same final concentration (1 g/l).

The growth and production responses of the cultures were similar to those obtained above, with about 90% yield of thiocolchicosides.

Example 8

One liter of ST broth in Erlenmeyer flasks was inoculated with a cryogenic tube culture of the DSM1670 strain. The flasks were incubated overnight at 30°C, 250 rpm. The inoculum was aseptically transferred to a 14 liter fermenter containing 9 liters of sterile ST broth to which thiocolchicine was added at a final concentration of 1 g/l. Keep suitable agitation-aeration level (stirring rate 900rpm; aeration 1-1.5vvm), complete fermentation. Every 2 hours, samples were taken from the culture broth and analyzed as follows:

Optical Density (OD) at -600nm

- Analysis of sterility and purity of the strains on LB agar;

- Microscopic morphology (Gram bacteria)

- Content analysis of thiocolchicine by TLC and HPLC.

After 28 hours of fermentation, conversion to thiocolchicosides was essentially complete. The final yield was about 90%. Example 9

The procedure described in Example 8 was repeated, but after 28 hours of fermentation, only 90% of the culture broth was recovered to extract the product (fraction 1). The remaining 10% was aseptically added to the fermenter containing 9 liters of fresh sterile ST medium containing 10 g of thiocolchicine. Fermentation was accomplished as described in Example 8. After 26 hours, 9 liters of culture broth were collected and extracted (fraction 2). Add the remaining volume of culture broth to 9 liters of sterile fresh ST medium containing thiocolchicine. After 26 hours, the culture broth was thoroughly collected and extracted (fraction 3). The biotransformation activity of the strain was stable in all 3 rounds with a conversion rate of about 90%, corresponding to essentially 3-fold the production of total thiocolchicosides compared to that obtained in a single batch process.

Example 10

The final broth of the fermentation (total volume: about 27 liters) was subjected to cross-flow microfiltration on a 0.22 μm ceramic cartridge to separate the cells from the broth. The permeate was adsorbed on a column packed with HP21, Mitsubishi adsorption resin. After washing with water, the product was eluted with methanol. The methanol eluate was concentrated to dryness in vacuo, then redissolved in methanol. After repeated extraction with dichloromethane, the alcoholic fraction was concentrated to dryness and redissolved in a 1:1 mixture of ethanol-dichloromethane. After clarification with silica gel, the solution was concentrated in vacuo; dichloromethane was then replaced with ethanol. The resulting suspension was concentrated and left to crystallize. After redissolving in an ethanol-chloroform mixture and clarifying on silica gel, a second crystallization from ethanol was carried out.

The obtained product was analyzed by HPLC, C-NMR, H-NMR and mass spectrometry, and it was proved to be the same as the standard of thiocolchicoside.

Claims (4)

1. the method for preparing formula (I) 3-O-glycosylcolchicine compound,

Wherein R ₁ is O-glycosidic group, R ₂ is hydrogen or C ₁ -C ₇ acyl, R ₃ is C ₁ -C ₆ alkoxy or C ₁ -C ₆ thioalkyl, the method comprises submerged culture, Compounds wherein R ₁ is OH or methoxy were biotransformed with Bacillus megaterium at 28-40°C, pH 6-7, using glucose, fructose or glycerol as carbon source. 2. The method of claim 1, preparing a compound of formula (I), wherein R ₁ is O-glucosidyl. 3. The method of claim 2 for the preparation of colchicosides and thiocolchicosides. 4. A method according to any one of claims 1-3, wherein the culture medium used contains 0.1-3 g/l of the compound of formula I wherein _R1 is hydroxy or methoxy.